Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B30/00—Methods of screening libraries
  • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• the present invention provides a method for evaluating, identifying, and analyzing the structure of a substance that interacts with a protein present in cells of an organism, and a substance that interacts with a specific protein in a cell of an organism. It relates to a method for determining the presence of a protein.
• Intracellularly In the cell surface or intracellularly (in this specification, both are sometimes simply referred to as “intracellularly"), there is a role as a receptor (sometimes referred to as “receptor” in this specification), and ion channels.
• the bioassay method using a protein whose activity is known has been common, but the bioassay method has at least two problems.
• MAL DI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
• capillary HPL CZMS and capillary HPL C / MS / MS online capillary reversed-phase high-performance liquid chromatography with mass spectrometric detection
• This tool provides excellent information on the molecular weight of the peptide in the sample, and the MS / MS format allows the peptide to fragment, generate cleavage ions, and deduce its amino acid sequence.
• the present inventors have already developed a method for directly identifying the chemical structure of a substance present in a biological tissue or cell using MALD I-TOF MS, and have filed an application as Japanese Patent Application No. 2000-060754.
• the present invention has been completed as a result of intensive research to expand the application range of the above-mentioned application.
• the present invention determines the presence of this protein or the presence of a substance that interacts with the protein by using a protein present in the cell membrane or in the cell and a substance that interacts with the protein.
• Fig. 1 is a chart showing the results of MALD I-TOF-MS measurement of the ligand (hANP) -bound membrane fraction prepared using cells expressing GC-A, a receptor for hANP, in Example 1. is there.
• FIG. 2 is a chart showing the results of MALD I-TOF-MS measurement of a ligand (hANP) -bound membrane fraction prepared using blank cells having no expression of GC-A in Example 1.
• Fig. 3 shows MALD I-TOF of the ligand-bound membrane fraction prepared using ligand (a peptide mixture consisting of four peptides containing hANP) and GC-A-expressing cells in Example 2. — A chart showing the results of MS measurement.
• Figure 4 shows MALD I-TOF-MS measurement of the ligand-bound membrane fraction prepared using the ligand (a peptide mixture consisting of four peptides containing hANP) and blank cells in Example 2. It is a chart showing the results.
• FIG. 5 is a chart showing the results of MALD I-TOF-MS measurement of only the peptide mixture used in Example 2.
• the cell when a known protein is present in a cell membrane or in a cell, the cell is brought into contact with an unknown test substance, and the test substance is adsorbed, bound, or associated with the cell. Interact in form.
• cells or cell sections for example, cell membranes
• the second embodiment of the method of the present invention uses a known substance, for example, a ligand for a specific receptor, and the presence of an unknown protein existing in a cell membrane or in a cell, contrary to the first embodiment.
• the cell containing the unknown protein is brought into contact with a known substance, and the known substance is adsorbed, bound, or associated with the cell.
• the known substance is adsorbed, bound, or associated with the cell.
• amino acid sequence of a protein whose presence has been confirmed can also be determined by the method of the present invention.
• ligands acting on various receptors expressed not only on the cell membrane but also on the nuclear membrane and the mitochondrial membrane are purified in a state of being adsorbed to the membrane, and the ligand-bound cell membrane is subjected to laser beam irradiation.
• laser beam irradiation only the ligand can be directly ionized, and the ligand can be analyzed by mass spectrometry.
• Receptors include so-called peptide hormone receptors, ion channels, transporters, and antibodies expressed on lymphocytes, macrophages, and the like.
• the ligand includes not only various natural physiologically active substances such as peptides, amino acids, amines and steroids but also synthetic compounds acting as ligands. Therefore, this method is effective as a means for searching for an unknown ligand that interacts with such a receptor, and can also be used for searching for a receptor whose function is unknown on a cell membrane.
• Mass spectrometry is used for analysis system, for example, if the cell number 1 0 4 -1 0 5 content of cell membrane fraction Cells, 2 mm X 2 mm thick if tissue sections 0. 1 mm ultrafine It can be analyzed with a large amount of sample, and in a short time, the molecular weight and the type of molecule (by peptide, protein, steroids, lignans, catechins, sugars, etc., hereinafter referred to as “chemical species”), and Information on molecules such as the structure of the molecules can be obtained at the same time. That is, if the MA LDI-TOF type is used as a mass spectrometer, the molecular weight of the ligand is measured. In addition, using the molecule-related ions as precursor ions / —co 1 umn LC-ESI—QTOF—MS / MS analyzer makes it possible to analyze the chemical species and structure of the adsorbed molecules. .
• the method of the present invention can be widely applied to not only a combination of a receptor and a ligand but also any substance that interacts with a protein in some form. These interactions include, when the protein is an ion channel, a transport protein, an antibody, an enzyme, or the like, an action on these. Substances having such an action include antigens for antibodies, substrates for enzymes, or activators or inhibitors.
• the present invention will be described by taking an example of detecting and identifying a ligand for a cell that expresses a known receptor.
• Known cells expressing a known receptor are cultured to express the receptor.
• the cultured cells are used as sample cells and treated as follows. For comparison, allogeneic cells not expressing the receptor were used.
• isolated cells such as tissue cells from organ tissues, lymphocytes, and monocytes in the abdominal cavity can also be used.
• Samples cells suitable number for example 1 0 3 to 1 0 5, preferably 1 0 4 to 1 0 qs five in distilled water, e.g. 1 0 0-8 0 0 1
• 3 at room temperature for 3 The cells are disrupted by incubating for 0 minutes, preferably for 5-20 minutes, and the cell membranes are sedimented by centrifugation or separated by filtration, washed with distilled water and centrifuged or filtered again. Then, the cytoplasm is removed to obtain a receptor-expressing cell membrane fraction.
• a substance that has the effect of preventing nonspecific adsorption of a substance sufficient to coat the nonspecific adsorption portion of the membrane fraction on the obtained membrane fraction for example, a 1 pmo 1Zz1 albumin (BSA) solution
• BSA 1 pmo 1Zz1 albumin
• a ligand for example, 10 pmo11 ligand solution
• 1001 of a ligand for example, 10 pmo11 ligand solution
• the mixture is added at room temperature for 3 to 30 minutes, preferably 5 to 30 minutes. Incubate for ⁇ 20 minutes. Then wash with distilled water Washing by washing and centrifugation (or filtration) is repeated twice or more, preferably 4 to 5 times, to remove excess ligand-unadsorbed fraction.
• distilled water for example, 5 to 201
• An appropriate amount of distilled water for example, 5 to 201, is added to the obtained ligand-adsorbed cell membrane, and suspended at Vo1tex. Dispense 0.5 to 31 of the suspension into a MALD I plate and air dry. Add ⁇ -cyano-14-hydroxycinnamic acid (aCHCA) matrix solution and measure MALD I-T ⁇ F-MS. By performing this measurement similarly for cell membranes that do not express a receptor for comparison and comparing the results, the ligand bound to a known receptor can be identified using the molecular weight as an index. The molecular ion associated with the laser used for the analysis was used as the precursor ion. — Co 1 umn LC— ES I—QTOF— MSZMS analyzer can be used to analyze the chemical species and structure of the adsorbed molecules. It is possible.
• a protein existing on the cell surface or in a cell or a substance interacting with the protein can be detected, identified or analyzed by the following steps.
• a substance interacting with a known protein existing on the cell surface or in a cell can be screened by a method comprising the following steps.
• test substance is a substance that interacts with the protein. Then, by identifying the substance, a substance that interacts with a known protein present on the cell surface or in the cell can be screened and identified.
• a cell or tissue expressing a receptor that interacts with a known ligand can be screened by the following method.
• the mass spectrum obtained by (1) was compared with the mass spectrum obtained by performing only the steps after (2) without contacting with the ligand of (1), and the former increased the amount of ligand. In such a case, it is a method of determining that the receptor interacting with the ligand is present in the tissue or the cell.
• Example 1 Binding experiment between human atrial natriuretic peptide (hereinafter referred to as "hANP”) and hANP receptor GC-A
• Cultured cells used were CHOZGC-A, a receptor expressing cell, ie, CHO cells (Chinese hamster ovary cells) expressing hANP receptor GC-A, and CHO host cells for blanks. F—MS Preparation was performed up to the measurement sample.
• albumin membrane fraction To the obtained albumin membrane fraction was added 10 1 pmo 11 hANP aqueous solution 100 1 and incubated at room temperature for 10 minutes. The suspension was centrifuged, and the obtained pellet was further washed with 5001 of distilled water and centrifuged again to obtain a ligand (hANP) -bound membrane fraction.
• hANP ligand
• hANP was detected about three times more in the GC-A expressing cells (FIG. 1) than in the blank cells (FIG. 2). From this, it can be determined that hANP interacts with GC-A receptor Yuichi.
• the albumin-coated membrane was prepared in the same manner as in Steps 1 and 2 of Example 1. (CHOZGC-A and CHO host cells).
• albumin membrane fraction a peptide mixture solution, i.e., hANP of 10 pmo11, angiotensin I of 10 pmo1 / 1, substance P (SP) of lOpmolZl and 10 pmo1 100/1 mixed aqueous solution of melanocyte stimulating hormone (K-MSH) in 1 / ⁇ 1 was added and incubated at room temperature for 10 minutes. The suspension was centrifuged, and the resulting pellet was further washed with 5001 of distilled water and centrifuged again to obtain a ligand-bound membrane fraction.
• hANP of 10 pmo11
• angiotensin I 10 pmo1 / 1
• SP substance P
• K-MSH melanocyte stimulating hormone
• MALD I—TOF—MS measurement was performed in the same manner as in step 4 of experiment 1. The results are shown in FIG. 3 (for the expression of the GC-A receptor) and in FIG. 4 (for the blank without the expression of the GC-A receptor). For comparison, peptide mixture (10 pmol / l hANP, 10 pmol / ⁇ 1 angiotensin I, 10 pmol / l SP, 10 pmo 1/1 ⁇ -MS H) 1 l ⁇ MALD I-TOF-MS measurement was performed for only 1 (Fig. 5).
• hANP bound to the GC-A receptor-expressing cell membrane fraction at least 5 times more selectively than the other three peptides. From this, it can be determined that only hANP interacts with the GC-A receptor, and other angiotensin I, substance P, and ⁇ -MSH do not interact.
• the cell when a known protein is present in a cell membrane or in a cell, the cell is brought into contact with an unknown test substance to allow the test substance to interact with the cell in any form such as adsorption, binding, or association. Let it work. In this state, cells or cell sections (eg, cell membranes) are purified, and the presence of the test substance is confirmed using MALD I-TOF MS to determine whether it interacts with a known protein. it can.
• cells or cell sections eg, cell membranes
• a known substance such as a ligand for a specific receptor
• the unknown substance existing in the cell membrane or in the cell is used.
• the protein on which the known substance acts is converted to a cell membrane or It can be determined that it exists in the cell.
• the amino acid sequence of a protein whose presence has been confirmed can also be determined by the method of the present invention.

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• 2002
  • 2002-06-19 JP JP2003505625A patent/JP4188229B2/ja not_active Expired - Fee Related
  • 2002-06-19 AT AT02738766T patent/ATE386937T1/de not_active IP Right Cessation
  • 2002-06-19 WO PCT/JP2002/006103 patent/WO2002103360A1/ja active IP Right Grant
  • 2002-06-19 DE DE60225140T patent/DE60225140T2/de not_active Expired - Lifetime
  • 2002-06-19 US US10/481,011 patent/US20040235062A1/en not_active Abandoned
  • 2002-06-19 EP EP02738766A patent/EP1406091B1/de not_active Expired - Lifetime

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